We are developing chemical and physical probes for the study of the mechanism of thiamine pyrophosphate and Mg(II) catalyzed reactions and initially testing these on yeast pyruvate decarboxylase. The proposed work includes (work in progress): 1. Determination of C12/C13 kinetic isotope effects on the enzyme and appropriate model reactions; b. Replacement of Mg(II) by Eu(III) and other lanthanides as well as by Mn(II) for Fluorescence and Nuclear Magnetic Resonance studies of the conformation of coenzyme bound to the enzyme; c. Development of fluorescent and photoaffinity labels for mapping the binding and catalytic sites; d. High resolution nmr on C13 and 'H nuclei of coenzyme and inhibitors when enzyme bound.